Interaction between the Natural Lipopeptide [Glu1, Asp5] Surfactin

Jan 25, 2010 - Fang Liu , Jingwen Xiao , Vasil M. Garamus , László Almásy , Regine Willumeit , Bozhong Mu , and Aihua Zou. Langmuir 2013 29 (34), 1064...
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Biomacromolecules 2010, 11, 593–599

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Interaction between the Natural Lipopeptide [Glu1, Asp5] Surfactin-C15 and Hemoglobin in Aqueous Solution Aihua Zou,†,‡ Jing Liu,† Vasil M. Garamus,† Kai Zheng,† Regine Willumeit,† and Bozhong Mu*,† State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, Shanghai 200237, PR China, and GKSS Research Center, Max-Planck-Str.1, 21502 Geesthacht, Germany Received October 7, 2009; Revised Manuscript Received November 28, 2009

The interaction between natural lipopeptide [Glu1, Asp5] surfactin-C15 (surfactin) and hemoglobin (Hb) has been studied. Surface tension measurements show that the critical micelle concentration (cmc) of surfactin increases from 1.54 × 10-5 to 3.86 × 10-5 mol/L with Hb. The UV spectra display that the effect of surfactin on Hb exhibits strong concentration-dependent fashion and the aquometHb convert to hemichrome at high surfactin concentration. Small-angle neutron scattering (SANS) and freeze-fracture transmission electron microscopy (FFTEM) measurements show that surfactin result in the formation of a fractal structure representing a “necklace model” of micelle-like clusters randomly distributed along the protein polypeptide chain at high surfactin concentration. Far-UV circular dichroism (CD) results confirmed that surfactin can disrupt the helical structure of protein at high concentrations, although the enhanced native-like behavior of protein by low concentration of surfactin was observed. The microenvironment change around Phe amino residues and disulfide bonds of Hb was obtained from near-UV CD spectra.

1. Introduction Surfactin, which is secreted by various strains of Bacillus subtilis, is one of the most powerful biosurfactants so far known. It consists of a peptide loop of seven amino acid residues (GluLeu-D-Leu-Val-Asp-D-Leu-Leu) and a C13-15 β-hydroxy hydrophobic fatty acid chain. Due to its amphiphilic structure, it can largely reduce the surface tension of water from 72 to 27 mN/m at the concentration of 1 × 10-5 mol/L.1,2 It is a strong foaming agent and a powerful emulsifier3,4 and exhibits a strong membrane destabilizing action. Besides the surface activities, surfactin has also shown ionophoric and sequestering properties.5,6 And more important is that surfactin exhibits significant biological activities, such as the antiviral,7,8 antibacterial,9,10 antiHIV,11 antitumor,12,13 and hemolytic13,14 activities. Compared with the chemically synthesized surfactants, surfactin is gaining an enormous interest because they are less toxic, more biodegradable, and diverse. All these properties make surfactin a very attractive compound for both industrial applications and biological studies. Hemoglobin (Hb), a kind of respiratory protein of vertebrate erythrocytes, is important in oxygen transport, H2O2 dispersion and electron transfer to all organs and parts of the body.15 It was also worth mentioning that hemichrome states of Hb are associated with blood abnormalities. Surfactant-protein interaction have been great interest in the fields of medicine, chemistry, biology, cosmetics, drug delivery,16,17 and so on. Alpert et al. showed that the intrinsic fluorescence of Hb primarily originates from the Trp and is sensitive to the R-T transition.18 The R form is the oxy/ligand bound form, while the T form is the deoxy form. The R and T forms show significant changes in relative fluorescence intensity.19 Venkatesh et al. examined that

Figure 1. Chemical structure of surfactin.

the heme released from reconstituted Hb in CTAB and SDS and they concluded that CTAB was capable of releasing heme better than SDS due to the stronger hydrophobicity of CTAB.20 In this work, the interaction between surfactin and Hb has been studied. Compared with conventional synthetic surfactant, surfactin has a large ring with seven amino acid residues, which may contribute strong hydrogen bonds to the interaction between surfactin and Hb. On the other hand, surfactin has strong hemolytic activity with red blood cells as reported. Thus, it is quite interesting to know the interaction between surfactin and Hb for the further study of surfactin’s hemolysis activity. In addition, surfactin is usually considered as more effective, more environmentally friendly, and more stable than many synthetic compounds. These aspects inspire us to explore whether surfactin has a special effect on Hb structure. To understand the interaction between surfactin and Hb, surface tension, UV-vis, SANS, and CD measurements were performed in this paper.

2. Materials and Methods * To whom correspondence should be addressed. Tel.: +86-21-64252063. Fax: +86-21-64252458. E-mail: [email protected]. † East China University of Science and Technology. ‡ GKSS Research Center.

2.1. Materials. The [Glu1, Asp5] surfactin-C15 isoform (Figure 1) consists of a heptapeptide headgroup with the sequence Glu-Leu-LeuVal-Asp-Leu-Leu closed to a lactone ring by a β-hydroxy fatty acid

10.1021/bm9011453  2010 American Chemical Society Published on Web 01/25/2010

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with 15 carbons. It was originally obtained from the cell free broth of Bacillus subtilis HSO121 in our laboratory.21,22 The separation process for the surfactin is done according to the references.23,24 These lipopeptides were separated by extraction with methanol, isolation with normal pressure ODS C18 column and purified by the RP-HPLC (Jasco, Japan). Then the structure of the isolated lipopeptide was determined by the electrospray ionization-time-of-flight mass spectrometer (ESITOF MS/MS) and GC/MS. During all the experiments, the samples were prepared with 0.01 mol/L phosphate buffer solution (PBS, pH 7.4). Bovine hemoglobin (Hb) was purchased from Sigma. Double distilled water was used for preparation of all solutions, except D2O was used for the sample preparation of SANS measurements. 2.2. Surface Tension Measurements. The surfactin was dissolved in 0.01 mol/L phosphate buffer solution (pH 7.4). Surface tension measurements were determined at 25.0 °C with a DCA 315 series system (Thermo-Cahn Instruments, Inc. U.S.A.). Prior to the measurement, the equipment was tested by determination of a surface tension σ ) 72.7 ( 0.2 mN/m of double distilled water at 25.0 °C. The surface tension versus ln(surfactant concentration) plots were used to determine cmc and σcmc. Origin 7.5 software was used to fit these curves and to determine the cmc, precmc slope, and surface tension of the concentration at which micelles are formed according to the function:

f(CS) ) -

∂σ ln cmc - ln(CS)g(CS) + σcmc ∂ ln CS

(1)

with g(CS) ) 1 for CS e cmc and g(CS) ) 0 for CS > cmc, where σcmc is the surface tension at surfactant concentrations higher than the cmc and ∂σ/∂ ln CS describes the alteration in the surface tension as a function of the natural logarithm of the surfactin concentration CS (slope). The cmc, σcmc, and ∂σ/∂ ln CS were used as fitting parameters in a least-squares fit. All values were reported as the mean and standard deviation according to the fitting curves. The excess surface concentration Γ can be calculated as25

Γ)-

(

∂σ 1 kRT ∂ ln(CS)

)

(2)

where σ is the surface tension [N/m], R is the gas constant, T is the temperature in [K], and CS is the surfactant concentration [mol/L]. In the case of ionic surfactants with two charges, counterions were considered by setting k to 3.25 From the maximum excess surface concentration, the area occupied by the molecule to micelle (A) in [Å2] was calculated by

A ) (Γ · L)-1

(3)

where L is Avogadro’s number. The free energy gain of micellization 0 (∆GM ) associated with micelle formation given by the relation26 0 ∆GM ) RT ln(cmc)

(4)

2.3. Small Angle Neutron Scattering (SANS). A total of 0.01 mol/L phosphate buffer (pD 7.4) was used to prepare the samples. SANS measurements were performed on the instrument SANS-1 at Geesthacht Neutron Facility GeNF, Geesthacht, Germany.27 To cover the range of scattering vectors q from 0.005 to 0.25 Å-1, four sampleto-detector distances between 0.7 m < d < 9.7 m were used. Samples were kept in quartz cells (Hellma) with a path length of 2 mm and placed in a thermostatted sample holder to ensure isothermal conditions of T ) 25.0 ( 0.5 °C. Raw data were corrected for background from the deuterium buffer solution and sample cell and other sources

Figure 2. Surface tension isotherms of surfactin solution with and without Hb (3.8 × 10-6 mol/L).

according to conventional procedures described in detail by Cotton.28 The two-dimensional isotropic scattering spectra were azimuthally averaged, converted to an absolute scale, and corrected for the detector efficiency by dividing by the incoherent scattering spectra of pure H2O, which was measured with a 1 mm path length quartz cell.29 The scattering functions were interpreted using scaling concepts. The simple interpretation was enhanced by numerical modeling to certain geometric shapes. The data were analyzed further by indirect Fourier transforms (IFT) by Glatter30 in approximation of spherelike objects (q > 0.02 Å-1) and trace scattering (q < 0.02 Å-1) from large aggregates. IFT is a model independent approach and requires only a minimum of prior information for analysis, that is, the maximum size and dimensions of aggregates (spherical, rod, or disk-like). The IFT method makes it possible to calculate the pair distance distribution function of scattering excess p(r) for spherical aggregates. From the p(r) function the radius gyration of the scattering length density excess Rg for spherical aggregates can be obtained.31 From the shape of the curve obtained by plotting p(r) against the distance in the micelles, the shape of the micelles can be estimated. A symmetrical shape is related to spheres. 2.4. Circular Dichroism (CD). The secondary structure of surfactin was probed by Jasco J-810 circular dichroism spectropolarimeter at 25 ( 0.1 °C. The far-UV and near-UV CD spectra of the surfactin at different concentrations were recorded with a spectral resolution of 0.1 nm. The scan speed was 100 nm /min and the bandwidth of 2 nm. Quartz cell with an optical path of 1 mm were used for far-UV CD spectra. While for near-UV CD spectra, the bandwidth of 1 nm and quartz cell with an optical path of 10 mm was conducted. The final spectra were obtained by subtracting the surfactin spectra from the related PBS buffer, then converting it to molar ellipticity (deg · cm2 · dmol-1) by its program. 2.5. Freeze-Fracture Transmission Electron Microscopy (FF-TEM). FF-TEM was used to characterize morphology of Hb/ surfactin aggregate structure in PBS solution (pH 7.4). Samples were immersed rapidly into the liquid ethane cooled by the liquid nitrogen. They were transferred into liquid nitrogen after about 5 s. The samples, after transferred into the chamber of the freeze-etching apparatus (BALZERS BAF-400D), were fractured at -120 °C, 3 × 10-7 mbar. After etched for 1 min, Pt-C was sprayed onto the fracture face at 45 °C, and then C was spayed at 90 °C. The replicas when taken out of the chamber were achieved at milli-Q water. Finally, the samples dispersed in PBS buffer solutions were loaded onto the TEM grids. The grids were examined at TEM (JEM-1400, JEDL. Tokyo, Japan).

3. Results and Discussions 3.1. Surface Tension Measurement and Geometric Properties. Figure 2 is the surface tension isotherm for pure surfactin, in which the critical micelle concentration (cmc) value of surfactin can be determined as 1.54 × 10-5 mol/L. The cmc value of 1.54 × 10-5 mol/L is relatively low in comparison

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Table 1. Data Calculated from the Pre-cmc Slope for Surfactin without and with Hb (3.8 × 10-6 mol/L) cmc (mol/L) surfactin 1.54 × 10–5 Hb/surfactin 3.86 × 10–5

σcmc (mN/m)

∂σ/∂ ln CS (mN/m)

A (Å2)

0 ∆GM (kJ/mol)

27.7 27.7

11.45 4.06

107.8 304.11

–27.5 –25.2

with that of other ionic surfactants, which shows that surfactin has a much stronger self-assembly ability. This result is similar with the results reported in references by Ishigami et al.1 and Han et al.32 With the addition of Hb (3.8 × 10-6 mol/L), the surface tension decreased obviously (Figure 2) at low surfactin concentration. This is because Hb molecules can decrease the surface tension due to its surface activities comparing to the water. However, the surface tension is higher than pure surfactin solution at the medium surfactin concentrations. The surface activity of pure surfactin decreases more rapidly due to the micelle formation in this region. For Hb/surfactin system, surfactin molecules bind onto the peptides chain of Hb and do not form micelles in the protein/surfactant complex. This phenomenon is similar with the surface tension properties of Myoglobin/SDS.33 Eventually, their surface tensions agree with the protein-free surfactin solution, suggesting that Hb molecules at the interface are gradually displaced by surfactin molecules due to competition in the adsorption layer. The cmc value of surfactin with 3.8 × 10-6 mol/L Hb increases to 3.86 × 10-5 mol/L when compared with the pure surfactin solution. Information obtained by fitting of the individual surface tension curves, including the cmc, surface tension above the cmc, the precmc slope, the area occupied by the molecule to micelle (A), and the free energy of micellization, are listed in Table 1. For pure surfactin solution, the calculation showed that area occupied by the molecule to micelle (A) is 107.8 Å2 and the free energy of micellization is -27.5 kJ/mol, which means that surfactin has a compact structure.34,35 In the presence of Hb, the cmc value of surfactin system increases, and the area occupied by the surfactin to micelle (A) increases from 107.8 Å2 to 304.1 Å2. In addition, the free energy of micellization decreases from -27.5 to -25.2 kJ/mol in the absence and presence of Hb. These results mean that the micellization activity of surfactin is less thermodynamically favorable with the addition of Hb, which is due to the interaction between surfactin and Hb. Thus, all the above results provide evidence of interaction between Hb and surfactin. And our results are similar with the surface tension changes of gemini surfactant by BSA addition.36 3.2. UV-vis Spectra. The Hb molecule contains four globin chains, of which two are R-chains and two are β-chains. Each chain contains the prosthetic group, that is, heme. In the center of each heme group is a Fe2+. The stability of the aquomet species is provided by the presence of a water molecule in the nonpolar pocket, which is stabilized by binding to the heme iron.37 The heme group is held in position by interaction with the histidine side chain of the globins.36,38 Figure 3 illustrates the UV-vis spectra of Hb (3.8 × 10-6 mol/L) in surfactin solutions. From Figure 3, it can be found that the UV-vis spectrum of Hb in PBS (Figure 3B) at pH 7.4 is same as the characteristic spectra of metHb with the characteristic peaks at 540 and 576 nm.39 Furthermore, due to the Q-band around 500 nm and the ligand-to-metal charge transfer (LMCT) band, approximately at 630 nm, which are spectral fingerprints of the aquometHb species, metHb used in

Figure 3. UV spectra of Hb (3.8 × 10-6 mol/L) with different surfactin concentrations.

the paper is an aquometHb species. Therefore, Hb exist mainly aquomet species in PBS solutions at pH 7.4. With the addition of surfactin, the absorption peak of aquomet molecules at 405 nm decreases due to the Soret absorption, and a red shift to 411 nm (Figure 3A). At the same time, there appears a new band at 535 nm (Figure 3B). The bands at 411 and 535 nm are considered to be characteristic of hemichrome complex. Therefore, surfactin can induce the aquometHb convert to hemichrome in PBS (pH 7.4). Here, it is noteworthy that the distinguished change of Hb UV-vis spectra happened when the surfactin concentration is higher than 3.0 × 10-5 mol/L (near to the surfactin’s cmc value). Thus, the surfactin concentration selected in the later SANS and FF-TEM measurements are higher than the cmc value of surfactin. It has been reported that UV-vis spectra of Hb are correlated with the interior of hydrophobic area around heme.40 Therefore, the different UV-vis spectra of Hb with and without surfactin are due to the interaction of surfactin molecule with Hb.37 Surfactin carries two negative charges at pH 7.4 because the pKa values of Asp and Glu are around 4.3 and 4.5, respectively. The isoelectric point (pI) of Hb is 6.86 and the pH of PBS buffer is 7.4. Thus, Hb has an overall negative charge slightly greater than zero and there exists an electrostatic repulsion between Hb and surfactin. In addition, there also exists the hydrophobic interaction and hydrogen bonds between Hb and surfactin. At low surfactin concentrations, the UV-vis spectra show that the aquometHb maintains its structure (Figure 3B) mainly due to the electrostatic repulsion between Hb and surfactin. While at high surfactin concentration (Figure 3B), the hydrophobic interaction and hydrogen bonds between Hb and surfactin destruct the hydrogen bond between the water ligand and the distal distidine. Thus, the water molecules can easily dissociate from the first coordination sphere of the iron to form hemichrome.41 Therefore, surfactin can induce aquometHb convert to hemichrome.42 The process contains two steps as

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Zou et al. Table 2. Results of SANS Data Analysis by a Model-Independent Approach (IFT) for Hb/Surfactin Solution Rg (Å)

I0 (cm-1)

24 ( 0.6 24 ( 0.33 23 ( 0.34 23 ( 0.35 21 ( 0.65

0.012 ( 0.0004 0.027 ( 0.0006 0.028 ( 0.0006 0.039 ( 0.0008 0.022 ( 0.0007

surfactin (mol/L) Hb (mol/L) Dmax (Å) 0 9.0 × 10-5 1.6 × 10-4 2.4 × 10-4 2.4 × 10-4

Figure 4. (A) Effect of the surfactin on SANS spectra of Hb (3.8 × 10-6 mol/L) in PBS; (B) P(r) function obtained from the corresponding scattering curves in A.

follows: the penetration of surfactin make Fe(II) exposed to oxidize into Fe(III) and the destruction of the hydrogen bond around heme. It is speculated that the hydrophobic parts of surfactin interact with some hydrophobic domain of Hb and subsequently facilitate iron oxidation.43 This phenomenon is similar with bacterial endotoxin.37,44 Similarly, the formation of hemichrome from metHb is also found to be concentrationdependent with SDS by Apurba et al.15 The stopped flow transient kinetic measurements of the interaction of SDS with metHb show that at least four molecules of SDS interact with one molecule of metHb. Liu et al. observed the formation of hemichrome below the critical micelle concentration (cmc) of surfactants (SDS and DTAB) and the release of heme from Hb above the cmc.42 3.3. Small-Angle Neutron Scattering. The effect of surfactin binding on Hb is illustrated by SANS spectra in Figure 4 with an Hb concentration of 3.8 × 10-6 mol/L in PBS. Our SANS experiments were started with free Hb in PBS buffer (Figure 4A). With the addition of surfactin, the scattering intensities of Hb were clearly increased especially at low q ( 0.02 Å-1) (Figure 4A).

-6

3.8 × 10 3.8 × 10-6 3.8 × 10-6 3.8 × 10-6

65 65 65 65 65

The pair distance distribution p(r) function (Figure 4B) also was obtained by applying the IFT method. The distance distribution function of Hb in PBS buffer exhibits a shape that is quite characteristic of an almost homogeneous locally spherical structure, and we obtain a first estimate of the diameter from the maximum distance of approximately 65 Å. This result is similar to those reported on Hb in literature.42 P(r) function (Figure 4B) showed that Hb/surfactin system also has some almost homogeneous shperelike aggregates. IFT analysis gave a the apparent radius of gyration of 24 ( 0.6 Å for Hb in PBS buffer (Figure 4B). And the apparent radius of gyration decreased to 23 ( 0.35 Å with the surfactin concentration at 2.4 × 10-4 mol/L (Table 2). So the apparent radius of gyration of Hb/surfactin system has no obvious change with the addition of surfactin. This result is conflicted with the surface tension and UV-vis spectral measurements (Table 2). About the above results from SANS, we suspected it may be due to the coexistence of Hb/surfactin mixture and surfactin self-aggregates. Figure 4A displays the distinguished change of the scattering intensities of Hb at low q ( 0.02 Å-1), the experimental data and the fitted curves coincide very well. Here, we got the estimation of the micelle diameter Dmax (Table 2), which is obtained from the maximum distance of p(r). After determination of the pair distance distribution function, the radius gyration Rg for spherical micelle was calculated (Table 2). It can be seen that the Rg value for surfactin solution is 21 ( 0.65 Å, which is smaller than the pure Hb system (24 ( 0.60 Å). The existence of small surfactin micelles is probably the reason of the unobvious change of the radius gyration Rg of Hb with the addition surfactin (Table 2). Figure 5 plots SANS data of pure surfactin solution as well as the fits to the IFT models with assumption of the trace contributions of large fractal objects (q < 0.02 Å-1). It can be seen that the curves for pure surfactin decay as ∼q-3.2 at low q. This result is different with the Hb/surfactin system with the same surfactin concentration, which decay as ∼q-2.8. Furthermore, the decay of pure surfactin solution falls within the limits of 3 and 4, which is an indication of fractal-like surface regions,46 characterized by fractal dimension for expected surface around 2.8. The above results point to a compact core of aggregates and fractal interface between aggregates formed by surfactin and the surrounding solvent. Therefore, surfactin

forms not only simple micelles but large aggregates as well even at rarely low concentration. Most probably, these are some sheets of surfactin bilayers, which, with a further increase of concentration, will form a lamellar phase. Here we stress that the fractal type scattering characteristics are not unique but could in principle arise also from specific type of size distribution of the objects. In summary, the difference obtained from Figure 5 both at low ( 0.02 Å-1) verified the interaction between surfactin and Hb. These results are strong supported by FF-TEM measurements, which were performed to observe the morphology change of Hb after the surfactin addition. The FF-TEM images of pure Hb, Hb/surfactin and pure surfactin were displayed in Figure 6. Figure 6A shows that the native Hb has a globe structure and the diameter is around 7 nm, which is well consistent with the SANS measurements. With the addition of surfactin, Figure 6B shows completely different morphology compared with Figure 6A, where appeared the small spherical, some necklace and some large irregular fracture aggregates. Here it should be noted that the necklace structure only appeared in the Hb/surfactin system, which is also reported in other protein/surfactant systems. Nuzhat et al. found that at high CTAB concentration, surfactant molecules result in the formation of a fractal structure representing a “necklace model” of micelle-like clusters randomly distributed along the polypeptide chain of BSA.47 Liu et al. found that the original structures of Hb became looser when CTAB concentration was higher than its critical micelle concentration. The Hb morphology has been changed or maybe denaturalized.48 Therefore, it can be argued that this kind of necklace structure in Hb/surfactin is related by the denaturation of protein and the binding of surfactin micelles on Hb, where the protein structure is almost fully unfolded. The morphology of surfactin aggregates at 4.0 × 10-5 mol/L was shown in Figure 6C, where different shapes of micelles are observed, and proves the existence of large aggregates. It is abnormal that at low surfactin concentration the bilayered unilamellar structure was found in Figure 6C (arrow) and also the wormlike micelles coexisted. Hence, surfactin has the strong self-aggregation property by an intramolecular hydrogen bond in PBS solution (pH 7.4), which is also verified by the above SANS measurements. This is a similar result from that obtained under more or less comparable conditions using electron cryomicroscopy by Knoblich et al.49 and using DLS measurements by Han et al.32 In addition, these results are also demonstrate our suspect that the coexistence of small spherical micelle formed by surfactin lead to the unobvious change of the radius gyration Rg of Hb with the addition surfactin (Table 2). 3.4. CD Spectra of Hb/Surfactin System. To understand the interaction process between surfactin and Hb, the CD experiments were conducted to study the Hb structural and conformational variation. Far-UV CD measurements were

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changes observed in the protein by far-UV and near-UV CD spectra suggesting native-like behavior at low surfactant concentration and unfolding at high concentration are found to be in tune with the results obtained from UV-vis spectra measurements.

4. Conclusions

Figure 7. Far-UV (A) and near-UV (B) CD spectra of Hb (3.8 × 10-6 mol/L) in different surfactin systems.

performed to monitor the secondary structure changes of Hb induced by surfactin (Figure 7A). The spectra were scanned in the wavelength region 200-240 nm to probe transition in backbone amide. The R-helix of protein is characterized by negative peaks of similar magnitude at 222 and 208 nm, respectively. At low surfactant concentrations (6.0 × 10-6 and 1.0 × 10-5 mol/L), the far-UV CD spectra of Hb solution have no obvious difference, even the R-helix of Hb seems a slight increase from that without surfactin. This result means that Hb maintains its natural secondary structure at low surfactin concentrations. With the increase of surfactin concentration, however, the R-helix of Hb is observed to decrease as seen from the amplitude decrease of the negative ellipticity at 222 and 208 nm. Apparently, the higher the added surfactin concentration, the less is the R-helix content, as observed previously. This is attributed to the binding of surfactant on protein surface, which leads protein to an extended disorder structure with exposed hydrophobic residues. Thus, we conclude that surfactant at a high concentration disrupts the helical structure of protein, and more solvent-exposed structures such as β-sheet and random coil appear. Near-UV CD measurements were also performed to monitor the third structure changes of Hb induced by surfactin (Figure 7B), which were scanned in the wavelength region 240-330 nm. It is well-known that the near-UV CD spectra of Hb is about the microenvironment around its aromatic amino residues like Trp, Tyr, Phe, and disulfide bonds. Figure 7B shows that the near-UV spectra of Hb has strong positive absorbance around 245-275 nm and weak absorbance 285-300 nm. With the addition of surfactin, the positive absorbance amplitude of Hb around 245-275 nm decreases at higher surfactin concentrations, while no obvious change around 280-300 nm. It is known that the absorbance of Phe amino residues of Hb is at 255, 261, and 268 nm. And the absorption of the disulfide bond is reflected almost the whole range of 250-320 nm. Therefore, the above results indicate that the interaction between surfactin and Hb leads to the microenvironment change around Phe amino residues and disulfide bonds. Therefore, the conformational

The combination of different analytical standard methods (surface tension, UV-vis spectra, SANS, and CD spectra) gave a comprehensive characterization of the biophysical properties of Hb in surfactin solution. UV-vis spectra show that the presence of surfactin can induce aquometHb convert to hemichrome and the effect of surfactin on Hb exhibits a strong concentration-dependent fashion, which also can be observed from CD measurements. At low surfactin concentrations, surfactin has no obvious effect on Hb structure, which is due to the electrostatic repulsion between Hb and surfactin and the high self-aggregation property of surfactin by an intramolecular hydrogen bond in PBS solution (pH 7.4). While at high surfactin concentrations, the R-helix of Hb is observed to decrease and the microenvironment change around Phe amino residues and disulfide bonds. These results are attributed to the binding of surfactant micelles on protein surface by hydrophobic interaction and intermolecular hydrogen bonds, which leads protein to an extended disorder structure with exposed hydrophobic residues. SANS and FF-TEM measurements further confirmed the above results and illustrated the morphological change of Hb with and without surfactin, where global structure and the necklace-like structure appeared in pure Hb and Hb/surfactin system, respectively. In conclusion, this study reveals that the structure of Hb can be effectively adjusted by a natural biosurfactant surfactin. Nature produced biosurfactant, surfactin, has interesting potential applications either as more sustainable substitutes for what we already use or in medicine such as antitumor and thrombus medicine. An important first step in applying them in either area is to understand how such molecules interact with the proteins in blood cells. This is the first study of the interaction of a naturally occurring biosurfactant surfactin with Hb, which could lead to new ideas for understanding surfactin’s hemolytic activity and also may assist in developing surfactin commercial formulations by avoiding its hemolytic activity and in widening their range of applications. Acknowledgment. A.Z. gratefully acknowledges the support of this work by the Alexander von Humboldt Foundation, the support of this work by Research Fund for the New Teacher of theDoctoralProgramofHigherEducationofChina(200802511024), the grant from the Ministry of Science and Technology of China (2007CB707801), and Shanghai Municipal Science and Technology Commission (071607014). We would like to thank ShuFeng Sun for making FF-TEM samples in the Center for Biological Electron Microscopy, the Institute of Biophysics.

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